Self-assembled particles from zwitterionic polymers and related methods

ABSTRACT

Zwitterionic block copolymers and zwitterionic conjugates that advantageously self-assemble into particles, particles assembled from the zwitterionic block copolymers and zwitterionic conjugates, pharmaceutical compositions that include the self-assembled particles, and methods for delivering therapeutic and diagnostic agents using the particles.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International Application No.PCT/US2010/055887, filed Nov. 8, 2010, which claims the benefit of U.S.Provisional Application No. 61/259,085, filed Nov. 6, 2009, each isexpressly incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under Contact No.N000140910137 awarded by the Office of Naval Research, Contract No. DMR0705907 awarded by the National Science Foundation, and Contract No. U54CA 119335-04S awarded by the National Cancer Institute. The Governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

The stability and targeting efficiency of nanoparticles (NPs) are thetwo most important issues for their applications to drug delivery anddiagnostic imaging. Coating materials are needed to render NPs bothstable and multi-functional to address these two issues. Polyethyleneglycol (PEG) is the material most commonly used to modify NPs forstabilization purposes due to its resistance to nonspecific proteinadsorption (or nonfouling properties). However, PEG is susceptible tooxidative damage and loss of function in biological media, which limitsits long-term applications. Besides their stability in complex media,the stability of NPs themselves is another important issue that is oftenoverlooked. NPs need to remain intact throughout any necessarymanufacturing processes such as centrifugation or lyophilization. Tomaintain the stability of NPs, including those coated with PEG, severalmeasures must be used such as low-speed ultrafiltration and addition ofcryoprotectants prior to freeze-drying. For targeting drug delivery,bio-recognition elements (e.g., targeting ligands) often need to beimmobilized onto NP surfaces. There is only one functional grouppotentially available at the end of a long PEG chain (e.g., 2-5 kDa) towhich to conjugate biomolecules. In addition, unreacted functionalgroups can cause non-specific binding, particularly in complex mediasuch as blood plasma and serum. With all current NP coating materials,one will have to compromise between excellent stability andmulti-functionality.

Despite the advances in the development of NP coating materials, a needexists for a single material or coating platform that can accommodateboth NP ultra-stability and multi-functionality.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a block copolymer, comprising:

(a) a zwitterionic polymer block comprising a poly(carboxybetaine), apoly(sulfobetaine) or a poly(phosphobetaine); and

(b) a hydrophobic block.

In one embodiment, the zwitterionic polymer block comprises a pluralityof repeating units, each repeating unit having the formula:

wherein

R₁ is selected from the group consisting of hydrogen, fluorine,trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups;

R₂ and R₃ are independently selected from the group consisting of alkyland aryl, or taken together with the nitrogen to which they are attachedform a cationic center;

L₁ is a linker that covalently couples the cationic center [N⁺(R₅)(R₆)]to the polymer backbone [—(CH₂—CR₄)_(n)—];

L₂ is a linker that covalently couples the anionic center [A(═O)—O⁻] tothe cationic center;

A is C, S, SO, P, or PO;

M⁺ is a counter ion associated with the (A=O)O⁻ anionic center;

X⁻ is a counter ion associated with the cationic center; and

n is an integer from 1 to about 10,000.

In another aspect, the invention provides a block copolymer, comprising:

(a) a mixed charge copolymer block comprising a mixed charge copolymer;and

(b) a hydrophobic block.

In one embodiment, the mixed charge copolymer comprises a plurality ofrepeating units, each repeating unit having the formula:

wherein

R₄ and R₅ are independently selected from hydrogen, fluorine,trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups;

R₆, R₇, and R₈ are independently selected from alkyl and aryl, or takentogether with the nitrogen to which they are attached form a cationiccenter;

A(═O)—OM) is an anionic center, wherein A is C, S, SO, P, or PO, and Mis a metal or organic counterion;

L₃ is a linker that covalently couples the cationic center[N⁺(R₆)(R₇)(R₈)] to the polymer backbone;

L₄ is a linker that covalently couples the anionic center [A(═O)—OM] tothe polymer backbone;

X⁻ is the counter ion associated with the cationic center;

n is an integer from 1 to about 10,000; and

p is an integer from 1 to about 10,000. For the above polymers, in oneembodiment, the hydrophobic block comprises a biocompatible polymer. Inone embodiment, the hydrophobic block comprises a homopolymer orcopolymer. In one embodiment, the hydrophobic block comprises a polymerselected from the group consisting of poly(lactic-co-glycolic acid),polycaprolactone, polyglycolide, polylactic acid,poly-3-hydroxybutyrate, polydioxanone, polytrimethylenecarbonate,poly(glycolide-co-caprolactone),poly(glycolide-co-trimethylenecarbonate), andpoly(dioxanon-co-trimethylenecarbonate-co-glycolide). In one embodiment,the hydrophobic block has a number average molecular weight from about1,000 to about 200,000.

In one aspect, the invention provides a zwitterionic polymer conjugate,comprising a lipid covalently coupled to a poly(carboxybetaine), apoly(sulfobetaine), or a poly(phosphobetaine).

In one embodiment, the poly(carboxybetaine), poly(sulfobetaine), orpoly(phosphobetaine) comprises a plurality of repeating units, eachrepeating unit having the formula:

wherein

R₁ is selected from the group consisting of hydrogen, fluorine,trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups;

R₂ and R₃ are independently selected from the group consisting of alkyland aryl, or taken together with the nitrogen to which they are attachedform a cationic center;

L₁ is a linker that covalently couples the cationic center [N⁺(R₅)(R₆)]to the polymer backbone [—(CH₂—CR₄)_(n)—];

L₂ is a linker that covalently couples the anionic center [A(═O)—O⁻] tothe cationic center;

A is C, S, SO, P, or PO;

M⁺ is a counter ion associated with the (A=O)O⁻ anionic center;

X⁻ is a counter ion associated with the cationic center; and

n is an integer from 1 to about 10,000.

In one aspect, the invention provides a mixed charge copolymerconjugate, comprising a lipid covalently coupled to mixed chargecopolymer.

In one embodiment, the mixed charge copolymer comprises a plurality ofrepeating units, each repeating unit having the formula:

wherein

R₄ and R₅ are independently selected from hydrogen, fluorine,trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups;

R₆, R₇, and R₈ are independently selected from alkyl and aryl, or takentogether with the nitrogen to which they are attached form a cationiccenter;

A(═O)—OM) is an anionic center, wherein A is C, S, SO, P, or PO, and Mis a metal or organic counterion;

L₃ is a linker that covalently couples the cationic center[N⁺(R₆)(R₇)(R₈)] to the polymer backbone;

L₄ is a linker that covalently couples the anionic center [A(═O)—OM] tothe polymer backbone;

X⁻ is the counter ion associated with the cationic center;

n is an integer from 1 to about 10,000; and

p is an integer from 1 to about 10,000.

For the above conjugates, in one embodiment, the lipid is adiacylphosphatidylethanolamine or a diacylphosphatidylglycerol. In oneembodiment, the lipid is selected from the group consisting ofdioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),16-O-monomethyl-phosphoethanolamine, 16-O-dimethyl-phosphoethanolamine,18-1-trans-phosphoethanolamine,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), and1,2-dioleoyl-sn-glycero-3-phophoethanolamine (transDOPE). In oneembodiment, the lipid is distearoyl-phosphatidylethanolamine (DSPE).

In other aspects, the invention provides particles.

In one embodiment, the invention provides a core-shell polymericparticle, comprising a plurality of block copolymers of the invention.

In one embodiment, the invention provides a micelle, comprising aplurality of conjugates of liposome, comprising a plurality ofconjugates of the invention.

In one embodiment, the invention provides a liposome, comprising aplurality of conjugates of the invention.

In one embodiment, the invention provides a polymersome, comprising aplurality of block copolymers of the invention.

The particles can further include one or more targeting agents, and oneor more therapeutic and/or one or more diagnostic agents.

In other aspects, the invention provides compositions that include oneor more of the particles of the invention and a pharmaceuticallyaccepted carrier or diluent.

In further aspects, the invention provides methods for delivering atherapeutic and/or diagnostic agent, comprising administering acomposition of the invention to a subject in need thereof.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings.

FIG. 1 is a schematic illustration of the preparation of arepresentative zwitterionic polymer conjugate of the invention, PLGA-PCBcopolymers; formation of PLGA-PCB/Dtxl NPs; and post-functionalizationof NPs with targeting ligands or diagnostic dyes.

FIGS. 2A-2F illustrate the characteristics of representativezwitterionic polymer conjugate nanoparticles. Scanning electronmicroscopy (SEM) images for PLGA-PCB NPs before (FIG. 2A) and after(FIG. 2B) lyophilization and brief re-suspension in water by pipetteswithout sonication. The scale bar is 1 μm. PLGA-PCB NP stability in PBSsolutions of 10 wt % BSA and in 100% FBS at 37° C. over 5 days (FIG.2C). NP size (Mean±SD, n=3) is plotted as a function of time. NPstability upon high-speed centrifugation for PLGA-PCB NPs (FIG. 2D) andPLGA-PCB/Dtxl NPs (1 wt % drug loading) (FIG. 2E) was tested with threesuccessive centrifugation cycles. After each step of centrifugation(16110 g, 15 min), supernatants were either removed or kept and the NPpellets were re-suspended by pipettes without sonication. Stability ofPLGA NPs, PLGA-PCB NPs, and PLGA-PCB/Dtxl NPs with 1 wt % drug loadingafter lyophilization without any addition of a cryoprotectant (FIG. 2F).NP size (Mean±SD, n=3) is plotted and the polydispersity indexes (PDIs,Mean±SD, n=3) accompanying each size point are indicated.

FIG. 3A illustrates the results of a studies on PLGA-PCB NPs conjugatedwith NH₂-Fluorescein. Bare NPs without fluorescein treatment, NPstreated with fluorescein (−EDC, +NHS), and NPs treated with fluorescein(+EDC, +NHS) are shown in black, blue, and green curves, respectively.NPs covalently bound with fluorescein (green lines) show a huge increaseof mean fluorescence intensity over NPs (−EDC+NHS+fluorescein). Bindingof PLGA-PCB/NBD NPs functionalized with galactose to HepG2 cells. Cellswere incubated for 2 h with PLGA-PCB/NBD NPs treated with NH₂-galactose(+EDC, +NHS) (FIG. 3B), and NH₂-galactose (−EDC, +NHS) (FIG. 3C).Fluorescence image and phase contrast image were taken at 20 h andcombined as illustrated in the figure.

FIG. 4 is a schematic illustration of the preparation a representativezwitterionic block copolymer of the invention, DSPC-PCB.

FIGS. 5A-5D compare size exclusion chromatography results as a functionof molar composition for representative particles (vesicles andmicelles) of the invention [DSPC/DSPE-PCB 5K (FIG. 5A); DSPC/DSPE-PCB 2K(FIG. 5B)] and related PEG particles [DSPC/DSPE-PEG 5K (FIG. 5C);DSPC/DSPE-PEG 2K (FIG. 5D)].

FIG. 6 compares liposome stability in PBS at 37° C. for a representativeparticle (liposome) of the invention (DSPC/DSPE-PCB 5K) to and relatedparticles.

FIG. 7 compares docetaxel release profiles from representativenanoparticles of the invention, PLGA-PCB NPs, with PLGA NPs. Drugloading for both NPs was 1 wt %.

FIGS. 8A and 8B compare NP stability (nanoparticle size) in 10 wt % BSAsolution in PBS (FIG. 8A), and 100% FBS solution at 37° C. (FIG. 8B). NPsize (mean±SD, n=3) was plotted as a function of time.

FIG. 9 compares cell viability (cytotoxicity) of representativenanoparticles of the invention, PLGA-PCB NPs, with PLGA NPs on HepG2cells. NPs are incubated with the cells at indicated concentrations for24 h, and immediately assayed for cell viability in triplicate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides block copolymers and conjugates thatadvantageously self-assemble into particles. The invention furtherincludes particles assembled from the block copolymers and conjugates,pharmaceutical compositions that include the self-assembled particles,and methods for delivering therapeutic and diagnostic agents using theparticles. Methods for making the block copolymers and conjugates andrelated particles are also provided.

Block Copolymers

In one aspect, the invention provides block copolymers. In oneembodiment, the block copolymer is a zwitterionic block copolymer. Inanother embodiment, the block copolymer is a mixed charge blockcopolymer

Zwitterionic Block Copolymer

In one embodiment, the invention provides a zwitterionic blockcopolymer. As used herein, the term “zwitterionic block copolymer”refers to a block copolymer having a zwitterionic polymer block.

In one embodiment, the block copolymer is a zwitterionic block copolymercomprising:

(a) a zwitterionic block comprising a poly(carboxybetaine), apoly(sulfobetaine), or a poly(phosphobetaine); and

(b) a hydrophobic block.

In one embodiment, the zwitterionic polymer block comprises a pluralityof repeating units, each repeating unit having the formula (I):

wherein

R₁ is selected from the group consisting of hydrogen, fluorine,trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups;

R₂ and R₃ are independently selected from the group consisting of alkyland aryl, or taken together with the nitrogen to which they are attachedform a cationic center;

L₁ is a linker that covalently couples the cationic center [N⁺(R₅)(R₆)]to the polymer backbone [—(CH₂—CR₄)_(n)—];

L₂ is a linker that covalently couples the anionic center [A(═O)—O⁻] tothe cationic center;

A is C, S, SO, P, or PO;

M⁺ is a counter ion associated with the (A=O)O⁻ anionic center;

X⁻ is a counter ion associated with the cationic center;

n is an integer from 1 to about 10,000; and

* represents the point at which the repeating unit is covalently linkedto the next.

Mixed Charge Block Copolymer

In one embodiment, the invention provides a mixed charge blockcopolymer. In one embodiment, the block copolymer is a mixed chargeblock copolymer comprising:

(a) a mixed charge copolymer block comprising a poly(carboxybetaine), apoly(sulfobetaine), or a poly(phosphobetaine); and

(b) a hydrophobic block.

As used herein, the term “mixed charge block copolymer” refers to ablock copolymer having a mixed charge polymer block.

As used herein, the term “mixed charge copolymer” refers to a copolymerhaving a polymer backbone, a plurality of positively charged repeatingunits, and a plurality of negatively charged repeating units. In thepractice of the invention, these copolymers may be prepared bypolymerization of an ion-pair comonomer.

The mixed charge copolymer includes a plurality of positively chargedrepeating units, and a plurality of negatively charged repeating units.In one embodiment, the mixed charge copolymer is substantiallyelectronically neutral. As used herein, the term “substantiallyelectronically neutral” refers to a copolymer that imparts advantageousnonfouling properties to the copolymer. In one embodiment, asubstantially electronically neutral copolymer is a copolymer having anet charge of substantially zero (i.e., a copolymer about the samenumber of positively charged repeating units and negatively chargedrepeating units). In one embodiment, the ratio of the number ofpositively charged repeating units to the number of the negativelycharged repeating units is from about 1:1.1 to about 1:0.5. In oneembodiment, the ratio of the number of positively charged repeatingunits to the number of the negatively charged repeating units is fromabout 1:1.1 to about 1:0.7. In one embodiment, the ratio of the numberof positively charged repeating units to the number of the negativelycharged repeating units is from about 1:1.1 to about 1:0.9.

Ion Pair Comonomers.

In one embodiment, the copolymers are prepared by copolymerization ofsuitable polymerizable ion pair comonomers.

Representative ion-pair comonomers useful in the invention have formulas(II) and (III):

CH₂═C(R₄)-L₃-N⁺(R₆)(R₇)(R₈)X⁻  (II)

CH₂═C(R₅)-L₄-A₂(═O)—OM  (III)

In this embodiment, the mixed charge copolymer has repeating unitshaving formula (IV):

wherein

R₄ and R₅ are independently selected from hydrogen, fluorine,trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups;

R₆, R₇, and R₈ are independently selected from alkyl and aryl, or takentogether with the nitrogen to which they are attached form a cationiccenter;

A(═O)—OM) is an anionic center, wherein A is C, S, SO, P, or PO, and Mis a metal or organic counterion;

L₃ is a linker that covalently couples the cationic center[N⁺(R₆)(R₇)(R₈)] to the polymer backbone;

L₄ is a linker that covalently couples the anionic center [A(═O)—OM] tothe polymer backbone;

X⁻ is the counter ion associated with the cationic center;

n is an integer from 1 to about 10,000;

p is an integer from 1 to about 10,000; and

* represents the point at which the repeating units is covalently linkedto the next.

In one embodiment, R₇ and R₈ are C1-C3 alkyl.

R₆, R₇, and R₈ are independently selected from alkyl and aryl, or takentogether with the nitrogen to which they are attached form a cationiccenter. In one embodiment, R₆, R₇, and R₈ are C1-C3 alkyl.

In certain embodiments, L₃ is selected from the group consisting of—C(═O)O—(CH₂)_(n)— and —C(═O)NH—(CH₂)_(n)—, wherein n is an integer from1 to 20. In certain embodiments, L₃ is —C(═O)O—(CH₂)_(n)—, wherein n is1-6.

In certain embodiments, L₄ is a C1-C20 alkylene chain. Representative L₄groups include —(CH₂)_(n)—, where n is 1-20 (e.g., 1, 3, or 5)

In certain embodiments, A is C or SO.

In certain embodiments, n is an integer from 5 to about 5,000.

In one embodiment, R₄, R₅, R₆, R₇, and R₈ are methyl, L₃ is—C(═O)O—(CH₂)₂—, and L₄ is —CH₂—, A₁ is C or SO, and n is an integerfrom 5 to about 5,000.

In the above formulas, the polymer backbones include vinyl backbones(i.e., —C(R′)(R″)—C(R′″)(R″″)—, where R′, R″, R′″, and R′″ areindependently selected from hydrogen, alkyl, and aryl) derived fromvinyl monomers (e.g., acrylate, methacrylate, acrylamide,methacrylamide, styrene).

In the above formulas, N⁺ is the cationic center. In certainembodiments, the cationic center is a quaternary ammonium (e.g., Nbonded to L₁, R₂, R₃, and L₂). In addition to ammonium, other usefulcationic centers (e.g., R₂ and R₃ taken together with N) includeimidazolium, triazaolium, pyridinium, morpholinium, oxazolidinium,pyrazinium, pyridazinium, pyrimidinium, piperazinium, and pyrrolidinium.

R₁-R₈ are independently selected from hydrogen, alkyl, and aryl groups.Representative alkyl groups include C1-C10 straight chain and branchedalkyl groups. In certain embodiments, the alkyl group is furthersubstituted with one of more substituents including, for example, anaryl group (e.g., —CH₂C₆H₅, benzyl). In one embodiment, R₂ and R₃, andR₆, R₇, and R₈, are methyl. In one embodiment, R₁-R₈ are methyl.Representative aryl groups include C6-C12 aryl groups including, forexample, phenyl. For certain embodiments of the above formulas, R₂ andR₃, and/or R₆, R₇, and R₈ are taken together with N⁺ form the cationiccenter.

L₁ is a linker that covalently couples the cationic center to thepolymer backbone. In certain embodiments, L₁ includes a functional group(e.g., ester or amide) that couples the remainder of L₁ to the polymerbackbone (or polymerizable moiety for the monomers). In addition to thefunctional group, L₁ can include an C1-C20 alkylene chain.Representative L₁ groups include —C(═O)O-—(CH₂)_(n)— and—C(═O)NH—(CH₂)_(n)—, where n is 1-20 (e.g., n=2).

L₂ is a linker that covalently couples the cationic center to theanionic group. L₂ can be a C1-C20 alkylene chain. Representative L₂groups include —(CH₂)_(n)—, where n is 1-20 (e.g., 1, 3, or 5).

L₃ is a linker that covalently couples the cationic center to thepolymer backbone. In certain embodiments, L₃ includes a functional group(e.g., ester or amide) that couples the remainder of L₃ to the polymerbackbone (or polymerizable moiety for the monomers). In addition to thefunctional group, L₃ can include an C1-C20 alkylene chain.Representative L₃ groups include —C(═O)O—(CH₂)_(n)— and—C(═O)NH—(CH₂)_(n)—, where n is 1-20 (e.g., n=2).

L₄ is a linker that covalently couples the anionic group to the polymerbackbone. L₄ can be a C1-C20 alkylene chain. Representative L₄ groupsinclude —(CH₂)_(n)—, where n is 1-20 (e.g., 1, 3, or 5).

Representative alkyl groups include C1-C30 straight chain and branchedalkyl groups. In certain embodiments, the alkyl group is furthersubstituted with one of more substituents including, for example, anaryl group (e.g., —CH₂C₆H₅, benzyl).

Representative aryl groups include C6-C12 aryl groups including, forexample, phenyl including substituted phenyl groups (e.g., benzoicacid).

X⁻ is the counter ion associated with the cationic center. The counterion can be the counter ion that results from the synthesis of thecationic polymers or the monomers (e.g., Cl⁻, Br⁻, I⁻). The counter ionthat is initially produced from the synthesis of the cationic center canalso be exchanged with other suitable counter ions. Representativehydrophobic counter ions include carboxylates, such as benzoic acid andfatty acid anions (e.g., CH₃(CH₂)_(n)CO₂ ⁻ where n=1-19); alkylsulfonates (e.g., CH₃(CH₂)_(n)SO₃ ⁻ where n=1-19); salicylate; lactate;bis(trifluoromethylsulfonyl)amide anion (N⁻(SO₂CF₃)₂); and derivativesthereof. Other counter ions also can be chosen from chloride, bromide,iodide, sulfate; nitrate; perchlorate (ClO₄); tetrafluoroborate (BF₄);hexafluorophosphate (PF₆); trifluoromethylsulfonate (SO₃CF₃); andderivatives thereof. Other suitable counter ions include salicylic acid(2-hydroxybenzoic acid), benzoate, and lactate.

For the zwitterionic polymers and mixed charge copolymers useful in theinvention, the degree of polymerization (DP or n), number averagemolecular weight (M_(n)), and the ratio of weight average and numberaverage molecular weights (M_(w)/M_(n).), also known as polydispersityindex, can vary. In one embodiment, the polymers have a degree ofpolymerization (n) from 1 to about 10,000. In one embodiment, n is fromabout 10 to about 5,000. In another embodiment, n is from about 100 toabout 3,500. In one embodiment, the polymers have a number averagemolecular weight (M_(n)) of from about 200 to about 2,000,000 Da. In oneembodiment, M_(n) is from about 2,000 to about 100,000 Da. In anotherembodiment, M_(n) is from about 20,000 to about 80,000 Da. In oneembodiment, the polymers have a ratio of weight average and numberaverage molecular weight (M_(w)/M_(n).) of from about 1.0 to about 2.0.In one embodiment, M_(w)/M_(n). is from about 1.1 to about 1.5. Inanother embodiment, M_(w)/M_(n). is from about 1.2 to about 2.0.

In the block copolymers, the hydrophobic block is the portion of thecopolymer that forms the core of the core-shell particle. Suitablehydrophobic blocks comprise a polymeric block that is biocompatiblepolymer. The hydrophobic block can be comprised of a homopolymer orcopolymer. The zwitterionic polymer block or the mixed charge copolymerblock form the shells of the particle.

Representative biodegradable hydrophobic blocks include peptides,polyesters, polyorthoesters, polyanhydrides, polyesteramides, andpolyoxaesters, and their derivatives or combinations thereof.

Representative hydrophobic blocks comprise a polymer selected frompoly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),polyglycolide (PG), polylactic acid (PLA), poly-3-hydroxybutyrate,polydioxanone, polytrimethylenecarbonate,poly(glycolide-co-caprolactone) (Monocryl™),poly(glycolide-co-trimethylenecarbonate) (Maxon™), andpoly(dioxanon-co-trimethylenecarbonate-co-glycolide) (BioSyn™).

In certain embodiments, the hydrophobic block has a number averagemolecular weight from about 1,000 to about 200,000 Da.

The block copolymers can be prepared by preparing a radical initiatorbased on the hydrophobic block (hydrophobic polymer functionalized toinclude a terminal radical initiator group) followed by polymerizationof a suitable carboxybetaine monomer. Alternatively, the blockcopolymers can be prepared by covalently coupling a suitablyfunctionalized hydrophobic polymer (e.g., end terminal amino group) to asuitably functionalized zwitterionic polymer (e.g., end terminal carboxygroup or reactive derivative thereof) or their hydrophobic derivatives(e.g., cationic ester derivatives).

Conjugates

In another aspect, the invention provides conjugates. In one embodiment,the conjugate is a zwitterionic polymer conjugate. In anotherembodiment, the conjugate is a mixed charge copolymer conjugate. Likethe block copolymers described above, the conjugates include ahydrophobic portion that comprises the core of the particle onself-assembly of the conjugate. The particle shell is comprised of thezwitterionic polymer portion or mixed charge copolymer portion.

Zwitterionic Conjugate

In one embodiment, the invention provides a zwitterionic polymerconjugate comprising a lipid covalently coupled to apoly(carboxybetaine), a poly(sulfobetaine) or a poly(phosphobetaine). Inanother embodiment, the invention provides a mixed charge copolymerconjugate comprising a lipid covalently coupled to apoly(carboxybetaine), a poly(sulfobetaine) or a poly(phosphobetaine).

Lipids suitable for use in the conjugates can be selected from a varietyof synthetic vesicle-forming lipids or naturally-occurringvesicle-forming lipids. These lipids include phospholipids,sphingolipids, and sterols. The lipid contain a chemical group such asamine group, hydroxyl group, aldehyde group, or carboxylic acid group atits polar head group suitable for covalent attachment of thezwitterionic polymer or mixed charge copolymer chains.

One embodiment includes two hydrocarbon chains, such asphosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidicacid (PA), or phosphatidylinositol (PI), where each hydrocarbon chaincontain 3-24 carbon atoms in length and have varying degrees ofunsaturation.

Suitable lipids include those derived fromdiacylphosphatidylethanolamines, ceramides, sphingomyelins,dihydrosphingomyelins, cephalins, and cerebrosides. For the diacylcompounds, the acyl group is a fatty acid group (e.g., C8-C40).

In certain embodiments, the lipid is a diacylphosphatidylethanolamine ora diacylphosphatidylglycerol.

Representative lipids include dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylethanolamine (POPE), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),16-O-monomethyl-phosphoethanolamine, 16-O-dimethyl-phosphoethanolamine,18-1-trans-phosphoethanolamine,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), and1,2-dioleoyl-sn-glycero-3-phophoethanolamine (transDOPE).

In one embodiment, the hydrophobic moiety isdistearoyl-phosphatidylethanolamine (DSPE).

For the zwitterionic polymer conjugates, the zwitterionic polymerportion of the conjugate is the same as described above for thezwitterionic polymer block (i.e., formula (I)).

For the mixed charge copolymer conjugates, the mixed charge copolymerportion of the conjugate is the same as described above for the mixedcharge copolymer block (i.e., formula (IV)).

The conjugates can be prepared by covalently coupling a suitablyfunctionalized hydrophobic moiety (e.g., end terminal amino) to asuitably functionalized zwitterionic polymer or mixed charge copolymer(e.g., end terminal carboxy group or reactive derivative thereof) ortheir hydrophobic derivatives (e.g., cationic ester derivatives).

The preparation and characteristics of representative zwitterionicconjugates of the invention, corresponding particles that include atherapeutic drug, and liposome formulations of the particles aredescribed in Example 1.

Particles

In another aspect of the invention, particles formed from thezwitterionic block copolymers and zwitterionic conjugates are provided.Because of the nature of the copolymers and conjugates, the particlescan be formed by self-assembly in aqueous environments. In an aqueousenvironment (e.g., physiological environment), the particles have ahydrophobic core comprising the hydrophobic portion of the copolymer,and a hydrophilic shell comprising the highly charged zwitterionicportion of the copolymer. In another embodiment, the particles have theform of a micelle composed of the conjugate.

In certain embodiments, the particles can have a vesicle structure. Inone embodiment, the particles have the form of a liposome, formulatedfrom the conjugate and other vesicle-forming lipids. Thesevesicle-forming lipids can be selected from a variety of syntheticvesicle-forming lipids or naturally-occurring vesicle-forming lipids.These lipids include phospholipids, sphingolipids, and sterols. Inanother embodiment, the particles have the form of a polymersome,formulated from the copolymers. For the delivery of cargo, such astherapeutic and/or diagnostic agents, core-shell nanoparticles andmicelles are suitable to encapsulated hydrophobic cargo, and liposomesand polymersomes prefer hydrophilic cargo, although hydrophobic cargocan also be encapsulated.

In another embodiment, the invention provides a particle comprising aplurality of zwitterionic conjugates of the invention. In certainembodiments, the particles further include one or more vesicle-forminglipids.

In one embodiment, the particle is a core-shell nanoparticle.

In one embodiment, the particle is a polymersome.

In one embodiment, the particle is a micelle.

In one embodiment, the particle is a liposome.

Core-shell nanoparticles and micelles are useful to coat hydrophobicparticles. Hydrophobic particles include metal particles (e.g., gold,silver, iron oxide, quantum dots) and polymeric particles.

In certain embodiments, the particles have a mean hydrodynamic diameterof from about 5 to about 5000 nm. In certain embodiments, the particleshave a mean hydrodynamic diameter of from about 5 to about 500 nm. Inother embodiments, the particles have a mean hydrodynamic diameter offrom about 5 to about 200 nm. Rigid NPs with diameter larger than kidneyglomerulus pore (about 5 nm) can effectively reduce renal filtration,thus increasing blood circulation time. Tumor vasculature has largerpore sizes than normal tissues (40-80 nm or even 1 μm), thus allowingpassive leakage of suitable sized NPs into tumors, which is called theenhanced permeation and retention (EPR) effect.

Therapeutic Agents.

In certain embodiments, the particles of the invention further includeone or more therapeutic agent. Exemplary therapeutic agents that may beused in accordance with the present invention include small molecules,organometallic compounds, nucleic acids, proteins (including multimericproteins, protein complexes, peptides), lipids, carbohydrates, hormones,metals, radioactive elements and compounds, drugs, vaccines,immunological agents, and/or combinations thereof.

In some embodiments, the therapeutic agent is a small molecule and/ororganic compound with pharmaceutical activity. In some embodiments, thetherapeutic agent is a clinically-used drug. In some embodiments, thedrug is an anti-cancer agent, antibiotic, anti-viral agent, anti-HIVagent, anti-parasite agent, anti-protozoal agent, anesthetic,anticoagulant, inhibitor of an enzyme, steroidal agent, steroidal ornon-steroidal anti-inflammatory agent, antihistamine, immunosuppressantagent, anti-neoplastic agent, antigen, vaccine, antibody, decongestant,sedative, opioid, analgesic, anti-pyretic, birth control agent, hormone,prostaglandin, progestational agent, anti-glaucoma agent, ophthalmicagent, anti-cholinergic, analgesic, anti-depressant, anti-psychotic,neurotoxin, hypnotic, tranquilizer, anti-convulsant, muscle relaxant,anti-Parkinson agent, anti-spasmodic, muscle contractant, channelblocker, miotic agent, anti-secretory agent, anti-thrombotic agent,anticoagulant, anti-cholinergic, β-adrenergic blocking agent, diuretic,cardiovascular active agent, vasoactive agent, vasodilating agent,anti-hypertensive agent, angiogenic agent, modulators ofcell-extracellular matrix interactions (e.g. cell growth inhibitors andanti-adhesion molecules), inhibitor of DNA, RNA, or protein synthesis.

In certain embodiments, a small molecule agent can be any drug. In someembodiments, the drug is one that has already been deemed safe andeffective for use in humans or animals by the appropriate governmentalagency or regulatory body. For example, drugs approved for human use arelisted by the FDA under 21 C.F.R. §§330.5, 331 through 361, and 440through 460, incorporated herein by reference; drugs for veterinary useare listed by the FDA under 21 C.F.R. §§500 through 589, incorporatedherein by reference. All listed drugs are considered acceptable for usein accordance with the present invention.

A more complete listing of classes and specific drugs suitable for usein the present invention may be found in Pharmaceutical Drugs:Syntheses, Patents, Applications by Axel Kleemann and Jurgen Engel,Thieme Medical Publishing, 1999 and the Merck Index: An Encyclopedia ofChemicals, Drugs and Biologicals, Ed. by Budavari et al, CRC Press,1996, both of which are incorporated herein by reference.

In certain embodiments of the invention, the therapeutic agent is anucleic acid (e.g., DNA, RNA, derivatives thereof). In some embodiments,the nucleic acid agent is a functional RNA. In general, a “functionalRNA” is an RNA that does not code for a protein but instead belongs to aclass of RNA molecules whose members characteristically possess one ormore different functions or activities within a cell. It will beappreciated that the relative activities of functional RNA moleculeshaving different sequences may differ and may depend at least in part onthe particular cell type in which the RNA is present. Thus the term“functional RNA” is used herein to refer to a class of RNA molecule andis not intended to imply that all members of the class will in factdisplay the activity characteristic of that class under any particularset of conditions. In some embodiments, functional RNAs includeRNAi-inducing entities (e.g., short interfering RNAs (siRNAs), shorthairpin RNAs (shRNAs), and microRNAs), ribozymes, tRNAs, rRNAs, RNAsuseful for triple helix formation.

In some embodiments, the nucleic acid agent is a vector. As used herein,the term “vector” refers to a nucleic acid molecule (typically, but notnecessarily, a DNA molecule) which can transport another nucleic acid towhich it has been linked. A vector can achieve extra-chromosomalreplication and/or expression of nucleic acids to which they are linkedin a host cell. In some embodiments, a vector can achieve integrationinto the genome of the host cell.

In some embodiments, vectors are used to direct protein and/or RNAexpression. In some embodiments, the protein and/or RNA to be expressedis not normally expressed by the cell. In some embodiments, the proteinand/or RNA to be expressed is normally expressed by the cell, but atlower levels than it is expressed when the vector has not been deliveredto the cell. In some embodiments, a vector directs expression of any ofthe functional RNAs described herein, such as RNAi-inducing entities,ribozymes.

In some embodiments, the therapeutic agent may be a protein or peptide.The terms “protein,” “polypeptide,” and “peptide” can be usedinterchangeably. In certain embodiments, peptides range from about 5 toabout 5000, 5 to about 1000, about 5 to about 750, about 5 to about 500,about 5 to about 250, about 5 to about 100, about 5 to about 75, about 5to about 50, about 5 to about 40, about 5 to about 30, about 5 to about25, about 5 to about 20, about 5 to about 15, or about 5 to about 10amino acids in size.

Polypeptides may contain L-amino acids, D-amino acids, or both and maycontain any of a variety of amino acid modifications or analogs known inthe art. Useful modifications include, e.g., terminal acetylation,amidation. In some embodiments, polypeptides may comprise natural aminoacids, unnatural amino acids, synthetic amino acids, and combinationsthereof, as described herein.

In some embodiments, the therapeutic agent may be a hormone,erythropoietin, insulin, cytokine, antigen for vaccination, growthfactor. In some embodiments, the therapeutic agent may be an antibodyand/or characteristic portion thereof. In some embodiments, antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric(i.e., “humanized”), or single chain (recombinant) antibodies. In someembodiments, antibodies may have reduced effector functions and/orbispecific molecules. In some embodiments, antibodies may include Fabfragments and/or fragments produced by a Fab expression library (e.g.Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, and Fd fragments).

In some embodiments, the therapeutic agent is a carbohydrate. In certainembodiments, the carbohydrate is a carbohydrate that is associated witha protein (e.g., glycoprotein, proteogycan). A carbohydrate may benatural or synthetic. A carbohydrate may also be a derivatized naturalcarbohydrate. In certain embodiments, a carbohydrate may be a simple orcomplex sugar. In certain embodiments, a carbohydrate is amonosaccharide, including but not limited to glucose, fructose,galactose, and ribose. In certain embodiments, a carbohydrate is adisaccharide, including but not limited to lactose, sucrose, maltose,trehalose, and cellobiose. In certain embodiments, a carbohydrate is apolysaccharide, including but not limited to cellulose, microcrystallinecellulose, hydroxypropyl methylcellulose (HPMC), methylcellulose (MC),dextrose, dextran, glycogen, xanthan gum, gellan gum, starch, andpullulan. In certain embodiments, a carbohydrate is a sugar alcohol,including but not limited to mannitol, sorbitol, xylitol, erythritol,malitol, and lactitol.

In some embodiments, the therapeutic agent is a lipid. In certainembodiments, the lipid is a lipid that is associated with a protein(e.g., lipoprotein). Exemplary lipids that may be used in accordancewith the present invention include, but are not limited to, oils, fattyacids, saturated fatty acid, unsaturated fatty acids, essential fattyacids, cis fatty acids, trans fatty acids, glycerides, monoglycerides,diglycerides, triglycerides, hormones, steroids (e.g., cholesterol, bileacids), vitamins (e.g., vitamin E), phospholipids, sphingolipids, andlipoproteins.

In some embodiments, the lipid may comprise one or more fatty acidgroups or salts thereof. In some embodiments, the fatty acid group maycomprise digestible, long chain (e.g., C8-C50), substituted orunsubstituted hydrocarbons. In some embodiments, the fatty acid groupmay be one or more of butyric, caproic, caprylic, capric, lauric,myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid. Insome embodiments, the fatty acid group may be one or more ofpalmitoleic, oleic, vaccenic, linoleic, alpha-linolenic, gamma-linoleic,arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic,or erucic acid.

Diagnostic Agents.

In certain embodiments, the particles of the invention further includeone or more diagnostic agents. In some embodiments, diagnostic agentsinclude commercially available imaging agents used in positron emissionstomography (PET), computer assisted tomography (CAT), single photonemission computerized tomography, x-ray, fluoroscopy, and magneticresonance imaging (MRI); anti-emetics; and contrast agents. Examples ofsuitable materials for use as contrast agents in MRI include gadoliniumchelates, as well as iron, magnesium, manganese, copper, and chromium.Examples of materials useful for CAT and x-ray imaging includeiodine-based materials.

In some embodiments, a diagnostic and/or therapeutic agent may be aradionuclide. Among the radionuclides used, gamma-emitters,positron-emitters, and X-ray emitters are suitable for diagnostic and/ortherapeutic purposes, while beta emitters and alpha-emitters may also beused for therapy. Suitable radionuclides for use in the inventioninclude, but are not limited to, 123I, 125I, 130I, 131I, 133I, 135I,47Sc, 72As, 72Se, 90Y, 88Y, 97Ru, 100Pd, 101mRh, 119Sb, 128Ba, 197Hg,211At, 212Bi, 212Pb, 109Pd, 111In, 67Ga, 68Ga, 67Cu, 75Br, 77Br, 99 mTc,14C, 13N, 15O, 32P, 33P, and 18F.

In some embodiments, a diagnostic agent may be a fluorescent,luminescent, or magnetic moiety. Fluorescent and luminescent moietiesinclude a variety of different organic or inorganic small moleculescommonly referred to as “dyes,” “labels,” or “indicators.” Examplesinclude fluorescein, rhodamine, acridine dyes, Alexa dyes, cyanine dyes.Fluorescent and luminescent moieties may include a variety of naturallyoccurring proteins and derivatives thereof, e.g., genetically engineeredvariants. For example, fluorescent proteins include green fluorescentprotein (GFP), enhanced GFP, red, blue, yellow, cyan, and sapphirefluorescent proteins, reef coral fluorescent protein. Luminescentproteins include luciferase, aequorin and derivatives thereof. Numerousfluorescent and luminescent dyes and proteins are known in the art (see,e.g., U.S. Patent Application Publication 2004/0067503; Valeur, B.,“Molecular Fluorescence: Principles and Applications,” John Wiley andSons, 2002; Handbook of Fluorescent Probes and Research Products,Molecular Probes, 9th edition, 2002; and The Handbook—A Guide toFluorescent Probes and Labeling Technologies, Invitrogen, 10th edition,available at the Invitrogen web site).

In some embodiments, a diagnostic agent may be nanoparticles that can bedetected by certain diagnostic methods, such as quantum dots, ironoxide, gold nanoparticles, nano-rod or nano-shell, carbon nanotube,nano-sheet, silica protected nanoparticles or combinations of thesenano-materials.

In certain embodiments, the particles of the invention further includeone or more therapeutic agent and/or one or more diagnostic agents.

Targeting Agents.

In certain embodiments, the particles (either with or without cargo)further include one or more targeting agents.

Particles useful for therapeutic and diagnostic purposes can beadvantageously treated with the polymers of the invention. In certainembodiments, the surface further comprises a plurality of target bindingpartners covalently coupled to a portion of the plurality of polymersadhered to the surface. In this embodiment, the target binding partnerhas affinity toward a target molecule. In these embodiments, thesurfaces can be used in diagnostic assays.

The binding affinity of a target molecule toward to the surface resultsfrom the target binding partners immobilized on the surface. The targetbinding partner and the target molecule, each termed a binding pairmember, form a binding pair. Each binding pair member is a molecule thatspecifically binds the other member. In one embodiment, the targetbinding partner has affinity to a target molecule with K_(d) less thanabout 10⁻⁸.

A binding pair member can be any suitable molecule including, withoutlimitation, proteins, peptides, proteins, poly- or oligo-saccharides,glycoproteins, lipids and lipoproteins, and nucleic acids, as well assynthetic organic or inorganic molecules having a defined bioactivity,such as an antibiotic, anti-inflammatory agent, or a cell adhesionmediator.

Examples of proteins that can be immobilized on the surfaces of thepresent invention include ligand-binding proteins, lectins, hormones,receptors, and enzymes. Representative proteins include antibodies(monoclonal, polyclonal, chimeric, single-chain or other recombinantforms), their protein/peptide antigens, protein-peptide hormones,streptavidin, avidin, protein A, proteins G, growth factors and theirrespective receptors, DNA-binding proteins, cell membrane receptors,endosomal membrane receptors, nuclear membrane receptors, neuronreceptors, visual receptors, and muscle cell receptors. Representativeoligonucleotides that can be immobilized on the surfaces of the presentinvention include DNA (genomic or cDNA), RNA, antisense, ribozymes, andexternal guide sequences for RNase P, and can range in size from shortoligonucleotide primers up to entire genes.

Other target binding partners that bind specifically to a targetcompound include poly- or oligosaccharides on glycoproteins that bind toreceptors, for example, the carbohydrate on the ligand for theinflammatory mediators P-selectin and E-selectin, and nucleic acidsequences that bind to complementary sequences, such as ribozymes,antisense, external guide sequences for RNase P, and aptamers.

In one embodiment, the target binding partner is an antibody, and thetarget molecule is an antigen against the antibody. In this embodiment,the surface of the invention specifically binds to the antigen andresists non-specific protein adsorption. In one embodiment, the targetbinding partner is a protein capable of promoting cell adhesion, and thetarget molecule is a cell. In this embodiment, the surface of theinvention specifically binds to the cell and resists non-specificprotein adsorption and non-specific cell adhesion.

The use of carboxybetaine polymer surfaces for immobilizing targetbinding partners is described in WO 2008/083390, expressly incorporatedherein by reference in its entirety.

Pharmaceutical Compositions

In other aspects, the invention provides compositions that include thezwitterionic block copolymers and zwitterionic conjugates. In certainembodiments, the compositions are pharmaceutical compositions suitablefor administration to subjects. These compositions includepharmaceutically accepted carriers or diluents.

Methods for Delivery of Therapeutic/Diagnostic Agents

In another aspect, the invention provides methods for using theparticles.

In one embodiment, the invention provides methods for delivering atherapeutic and/or diagnostic agent to a subject in need of therapy ordiagnosis. In the method, a composition comprising particles of one ormore of the zwitterionic block copolymers and zwitterionic conjugates ofthe invention are administered to the subject.

The following is a description of representative zwitterionic blockcopolymers and zwitterionic conjugates, their particles, and thecharacteristics thereof.

In one embodiment, the present invention provides a zwitterionicmaterial, poly(carboxybetaine) (PCB) that is unique in that each CB sidechain has one carboxylate anion group for conjugation withamine-containing biomolecules while at the same time each carboxylateanion group is paired with one cationic quaternary amine group as azwitterionic group to effectively resist non-specific protein adsorptioneven from complex media. The conjugation with biomolecules can be easilyachieved via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide andN-hydroxysuccinimide (EDC/NHS) chemistry. After conjugation, unreactedNHS ester groups are hydrolyzed to carboxylate anions, which are pairedwith cationic quaternary amines to form nonfouling zwitterionicstructures. However, for functionalizable COOH-terminated PEG, unreactedfunctional groups (e.g., carboxylate acid) can cause severe foulingproblems, particularly in complex media. Thus, PCB has abundantfunctional groups and an ultra low fouling background all in onematerial, which does not compromise the two quite different properties:nonfouling and functionalization. When antibodies are immobilized on aPCB-coated sensor chips, the post-functionalized surface still maintainsultra-low fouling properties (e.g., <0.3 ng/cm² adsorbed protein evenfrom undiluted blood plasma and serum) and biomarkers in blood can bedetected with high sensitivity. Introduction of CB onto NP surfacesimproves both stability and multi-functional abilities of drug deliverycarriers.

In one aspect, the present invention provides a poly(lactic-co-glycolicacid) (PLGA)-based drug delivery system. PLGA, approved by the FDA, hasbeen used to encapsulate and control drug release due to its hydrophobicand slow-hydrolysis nature in aqueous medium. In one embodiment, theinvention provides zwitterionic block copolymers (e.g., PLGA-PCB blockcopolymers) that self-assemble into PLGA-core NPs with a PCB-shell fordrug delivery.

PLGA-PCB NPs exhibit stabilizing effects due to the strong hydration ofzwitterionic CB and the sharp hydrophilicity/hydrophobicity differencebetween the PLGA-core and the PCB-shell. This difference between twoblocks is so great that no common solvents or mixed solvents canco-dissolve PLGA and PCB homopolymers. Thus, the synthesis of PLGA-PCBblock copolymers is very challenging due to the solvent problem. Tosolve this issue, a new carboxybetaine tert-butyl (CB-tBu) ester monomerwas prepared (see FIG. 1). Unlike zwitterionic CB, CB-tBu is a cationicester monomer with solubility in organic solvents, thus enabling thecovalent binding of PLGA with PCB-tBu polymers in a common solvent, suchas acetonitrile. Zwitterionic CB structures can be regenerated byhydrolysis of the tBu ester groups in an acid environment, such astrifluoroacetic acid (TFA), after covalent bonding with PLGA. Thissynthetic route potentially broadens the applicability of zwitterionicCB molecules in organic synthesis, making reactions between polarzwitterionic CB and a wide range of hydrophobic molecules possible.

PLGA-PCB block copolymers may be prepared either via PLGA (withappropriate terminal group) initiated radical polymerization of CB orvia conjugation of PLGA and PCB (e.g., COOH terminated-PLGA couples withNH₂ terminated-PCB). The latter route is preferred because the NH₂-PCBproducts can be conjugated to a wide range of COOH-terminated molecules,including the commercially available PLGA, potential chemo-drugs, andproteins or enzymes for stabilization and multifunctional purposes.However, direct conjugation of NH₂-PCB to COOH-PLGA blocks is difficultdue to their dramatic difference in polarity of the two blocks.Zwitterionic PCB can only be dissolved in water or methanol, while PLGAcannot be dissolved in either solvent. PLGA is also known to behydrolyzed by trace amounts of water. To solve these “solvent” problems,the invention provides a CB-tBu ester monomer (FIG. 1), which is stableand has solubility in anhydrous organic solvents such as acetonitrileand DMF. CB-tBu ester monomers are polymerized via an atom transferradical polymerization (ATRP) method initiated by a TFA⁻NH₃ ⁺-bearinginitiator (2-aminoethyl 2-bromoisobutyrate) (FIG. 1, Step 1). Afterremoval of the TFA salt, PCB-tBu-NH₂ with good solubility in organicsolvents is obtained (FIG. 1, Step 2) enabling conjugation with PLGA-NHSin anhydrous acetonitrile to form PLGA-PCB-tBu block copolymers (FIG. 1,Step 3). The uniqueness of the tBu ester lies in the easy removal of theester group by TFA to generate a zwitterionic CB structure while PLGAremains intact in such acidic environment (FIG. 1, Step 4). A 1-hour TFAtreatment is sufficient to fully convert PCB-tBu to PCB without breakingthe ester bond in its methylacrylate moiety, while up to 6 hours of TFAincubation will not destroy the ester backbone of PLGA. The resistanceof PLGA and polylactic acid (PLA) to acid degradation is known. Thus,PCB-tBu can be used in a generic way for the synthesis of anyamphiphilic block polymers containing one PCB block and anotherhydrophobic block, even though the hydrophobic part is subject tohydrolysis (e.g., PLA-PCB or PLGA-PCB).

To formulate PLGA-PCB NPs, a solvent displacement method (ornanoprecipitation method) was used in which the block copolymer isdissolved in a water-miscible organic solvent. Upon addition of water,in which the PCB block is soluble but PLGA block is not, “PLGA core-PCBshell” structured NPs form. The organic solvent is evaporated whilestirring, leaving an aqueous solution in which NPs harden. Whenhydrophobic drugs are mixed with copolymers in the organic solvent, theybecome encapsulated in the hydrophobic core of the NP during the processdescribed above (FIG. 1, Step 5). An organic solvent can play a decisiverole in NP formation due to the sharp difference in polarity betweenPLGA and PCB blocks. A single solvent, such as 2,2,2-trifluoroethanol(TFE), can dissolve PLGA-PCB largely due to the solubility of PLGA inthis solvent, but produces large particles with heterogeneous sizedistributions (515.8±50.0 nm, PDI=0.527±0.056). This results from themicroscopic insoluble state of PCB blocks in TFE. Thus, the formation ofinverted “PCB core-PLGA shell” micelles does not favor small andhomogeneous NPs. To improve the solubility of PCB block in TFE, aTFE/MeOH co-solvent was used instead, and NPs with a monodisperse sizedistribution, e.g., NP size=148.8±1.1 nm; PDI=0.040±0.011 (FIG. 2A),were then fabricated in a reproducible way. Note that TFE is preferredover DMSO due to the easy evaporation of TFE upon stirring. For atypical formulation, PLGA-PCB copolymers self-assembled into NPs withvery low PDI. The yield was nearly 100%, with no precipitates ormicro-size particles formed via the assembly process. Thus, there was noneed to use filtration to remove large particles. No surfactant isrequired during solvent displacement since zwitterionic PCB shellsstabilize the NPs in aqueous medium. Compared with PEGylated NP systems,PCB has better stabilization effect on NP dispersion because PCB is farmore hydrophilic than PEG, thus less chain embedment by PLGA is expectedduring the self-assembly process. The sharp polarity difference betweenthe copolymer blocks is responsible for such efficient assembly intosmall and homogeneous NPs. The zeta-potential for PLGA-PCB NPs obtainedabove was measured to be −43.5±1.0 mV, while PLGA NPs with the same size(NP size=145.7±3.9 nm; PDI=0.113±0.033) had a zeta-potential of−68.1±1.8 mV.

Docetaxel was encapsulated into the PLGA-PCB NPs via the method above,and the drug release profile was collected. For an initial drug input of5 wt % in the formulation, the resulting PLGA-PCB/Dtxl NPs had about 1wt % (0.933±0.021 wt %) drug load, a size of 138.5±0.6 nm, PDI of0.125±0.017, and zeta potential of −34.8±1.3 mV. PCB-modified andunmodified-PLGA NPs have similar sustained drug releasing profiles over96 hours with 50% of the encapsulated drug released in the first 8hours. Drug release kinetics can be further tuned by changing the lengthof the PLGA blocks; the rate of drug releasing can be prolonged byincreasing the molecular weight of PLGA.

The stability of NPs in biologically relevant media such as serumdetermines their feasibility as drug delivery vehicles for in vivo use.PCB, which has been found to effectively reduce non-specific proteinbinding on surfaces from undiluted blood plasma and serum, can stabilizehydrophobic PLGA NPs in complex media. PLGA-PCB NPs were placed in PBSsolution of 10 wt % bovine serum albumin (BSA) or 100% FBS solution at37° C. and the NP size was measured as a function of time. No sizeincrease of PCB-modified NPs was observed during the 13-hour study,while unmodified PLGA NPs severely aggregated immediately after theirimmersion in these media. A long-term study of PLGA-PCB NPs shows thatthese particles maintain their original size over a 5-day period in both10 wt % BSA and 100% FBS media (FIG. 2C). This implies that PLGA-PCB NPscan be used for in vivo drug delivery.

NP stability upon post-formulation or processing is also important fromNPs created in a laboratory to their clinical use. Because PCB bindswater through electrostatically induced hydration, it has strongerhydration than hydrogen-bonding materials such as PEG. It is expectedthat zwitterionic PCB polymers protect NPs from the harsh conditions ofvarious steps of processing. High-speed centrifugation is widely used toform NP pellets for purification purposes, but PLGA-based NPs, includingPEGylated NPs, tend to aggregate during pellet formation. PCB-modifiedPLGA NPs with or without drugs loaded were easily recovered fromrepeated high-speed centrifugation by brief pipette re-suspension of thepellets, thus abrogating the need for sonication. Pellets of unmodifiedPLGA NPs, once formed, however, cannot be resuspended withoutsonication. Specifically for PCB-modified NPs, when supernatants wereremoved at each centrifugation step (as in standard NP purificationprocedures), 6-9% and 1-8% increases in sizes were observed for PLGA-PCBNPs and PLGA-PCB/Dtxl NPs calculated from FIGS. 2D and 2E, respectively,with a slight decrease in PDI. The smaller PDIs obtained after eachcycle do reflect the monodispersity of those remaining NPs. Note thatthere is a slight size increase after each cycle which is not caused byNP aggregation. Smaller NPs are less likely to form the pellet at eachcentrifugation step, and thus the removal of supernatants will shift thesize distribution of the recovered portion towards larger values. Todouble confirm this stability issue, parallel experiments were performedin which the supernatant was not removed between centrifugation steps.It was found that NPs maintained the same size and PDI upon repeatedcentrifugation, indicating the ability of PCB to stabilize PLGA NPs(either with drug encapsulated or not) from any aggregation uponpelleting. This is due to the strong hydration layer created by the PCBshells of the NPs, stabilizing the hydrophobic PLGA cores frommechanically-induced aggregation.

Freeze-drying is a necessary procedure for NP storage to prevent polymerdegradation and drug leakage in aqueous storing media. No otherpolymer-based NPs can survive lyophilization without cryoprotectantadditives. Even for PEGylated NPs, additives such as 10% sucrose arerequired, because PEG is crystallized upon freeze-drying and loses itsfunction to prevent NP aggregation. The PCB-modified PLGA NPs retaintheir stability after freeze-drying without any additives (FIG. 2F). Abrief re-suspension with pipettes (no sonication needed) of the dryPCB-modified NPs either with or without drug loaded recovers the NPs tothe same mean diameter and low PDIs. These NPs were also visualizedafter freeze-drying by SEM (FIG. 2B). This behavior may result fromstrong PCB hydration and distinct PLGA/PCB blocks. Unlike PEG, PCBstrongly binds a certain amount of water molecules to preventcrystallization during lyophilization. In addition, the efficientassembly of the copolymers due to the sharp polarity contrast betweentwo blocks renders an almost defect-free protecting PCB shells keepingthe hydrophobic cores apart even in a highly dehydrated environment. Dueto their ability to survive in complex biological media and harshpost-formulation processing, zwitterionic PCB-modified NPs uniquelyaddress both industrial and clinical stability concerns.

Functionalization of NPs is required to attach biomolecules such as dyesand targeting ligands for different purposes. To examine the feasibilityof immobilizing those molecules of interest on PCB shells, fluoresceinwas used as a model ligand to generate fluorescent NPs. The carboxylategroups of PCB was converted to NHS ester in the presence of (+) EDC andNHS and later conjugated with amine groups in the fluorescein molecules.To confirm the conjugation chemistry on NP surfaces, NPs were preparedwithout generating NHS esters in the absence of (−) EDC but +NHS as thenegative control. Any binding of the dyes onto NPs in the negativecontrol should be due to physical interactions. After fluoresceinincubation, NPs with NHS esters on the surfaces had significantly higherfluorescence intensity than controls due to their covalent coupling withthe dyes (FIG. 3A). The potential of PLGA-PCB NPs as targeting drugdelivery vehicles is also evaluated. A green fluorescent dye (NBD) wasused as a “visible” model drug and encapsulated in the NPs. Theresulting PLGA-PCB/NBD NPs were further conjugated with amine-modifiedgalactose ligands with either +EDC and +NHS, or −EDC and +NHS, and thenwere incubated with HepG2 cells to test cell binding abilities.Galactose is widely used to target asialoglycoprotein receptors inhepatoma cell lines (e.g., HepG2) in vitro and hepatocytes in vivo.PLGA-PCB/NBD NPs with immobilized galactose ligands (+EDC, +NHS) readilybound to the cells, thus producing strong fluorescence as shown in FIG.3B. PLGA-PCB/NBD NPs without the immobilized ligands (−EDC, +NHS)resulted in non-targeting and nonfouling NPs and thus had low cellbinding abilities (FIG. 3C). These fluorescein and galactose/NBD resultsshow that PCB-modified NPs can be easily functionalized with amineterminating molecules for imaging and/or targeting purposes.

The structure of CB is similar to that of glycine betaine, a solutewhich is vital to osmotic regulation of living organisms. Estimates ofglycine betaine intake by humans are from 0.1 to 2.5 g/day. Thusmodification of PLGA NPs with biomimetic PCB should not bring anytoxicity to FDA-approved PLGA. Indeed, a cytotoxicity assay showed thatPLGA-PCB NPs were similar to PLGA NPs in terms of the viability of HepG2cells after 24 h incubation at NP concentrations up to 10 mg/ml. Thisconcentration corresponds to over 500 mg/kg body weight for an adulthuman, which is a much higher dose required for in vivo drug delivery.

The preparation and characteristics of a representative nanoparticledrug delivery system of the invention are described in Example 2. FIG. 7compares docetaxel release profiles from representative nanoparticles ofthe invention, PLGA-PCB NPs, with PLGA NPs. Drug loading for both NPswas 1 wt %. PCB modification did not change drug releasing behavior muchover unmodified PLGA. FIGS. 8A and 8B compare NP stability (nanoparticlesize) in 10 wt % BSA solution in PBS (FIG. 8A), and 100% FBS solution at37° C. (FIG. 8B). NP size (mean±SD, n=3) was plotted as a function oftime. FIG. 9 compares cell viability (cytotoxicity) of representativenanoparticles of the invention, PLGA-PCB NPs, with PLGA NPs on HepG2cells. NPs are incubated with the cells at indicated concentrations for24 h, and immediately assayed for cell viability in triplicate. Similarto PLGA NPs, PLGA-PCB NPs exhibit no cytotoxicity at concentrations upto 10 mg/ml.

PCB-modified NPs are superior over PEGylated NPs for their easyprocessing, extraordinary stability, and non-compromisedmulti-functionality. The abundant carboxylate anion groups of PCB enablethe attachment of targeting ligands, therapeutic drugs, and diagnosticlabels all in one material through conventional NHS/EDC chemistry,making PCB a universal platform for “theranostics”.

The following example are provided for the purpose of illustrating, notlimiting, the invention.

Example 1 Preparation and Characteristics of a RepresentativeZwitterionic Conjugate: DSPE-PCB

In this example, the preparation and characteristics of a representativezwitterionic conjugate of the invention, DSPE-PCB, and related liposomesare described. The preparation of the conjugate is illustrated in FIG.4.

DSPE-PCB Conjugate

NHS Ester Initiators (N-hydroxysuccinimide 2-bromopropanoate) for AtomTransfer Radical Polymerization (ATRP).

N-Hydroxysuccinimide (2.26 G, 19.6 Mmol) and 2-bromopropionic acid (1.45ml, 16.4 mmol) were dissolved in 500 ml of anhydrous dichloromethane ina round-bottomed flask, with a magnetic stirrer. The flask was cooled to0° C. and a solution of N,N′-dicyclohexylcarbodiimide (3.35 g, 16.34mmol) in dichloromethane (25 ml) was added dropwise. After stirring atroom temperature overnight the reaction mixture was filtered and thesolvent removed under reduced pressure to give a yellow solid. Theproduct was further purified by flash chromatography. Obtained 2.4 g ofwhite solid (9.63 mmol, yield=59%). ¹H NMR (chloroform) δ (ppm): 1.97and 2.00 (d, 3H, —COOCH(CH₃)Br), 2.89 (s, 4H, —COCH₂CH₂CO—), 4.64 (guar,J=6 Hz, 1H, —COOCH(CH₃)Br).

2-tert-Butoxy-N-(2-(methacryloyloxy)ethyl)-N,N-dimethyl-2-oxoethanaminium(CB-tBu)

5 g 2-(Dimethylamino)ethyl methacrylate and 8.68 g tert-butylbromoacetate were reacted in 20 ml acetonitrile for 24 h at 50° C. underN₂ protection. Upon addition of 250 ml ethyl ether to the reactionmixture, the formed white crystals were isolated and dried. The resultngCB-tBu monomers were immediately stored in a dessicator at −20° C.(yield 96%). ¹H NMR (D₂O) δ (ppm): 1.44 (s, 9H, —OC(CH₃)₃), 1.87 (s, 3H,CH₂═C(CH₃)COO—), 3.31 (s, 6H, —CH₂N(CH₃)₂CH₂COO—), 3.98 (t, J=3 Hz, 2H,CH₂═C(CH₃)COOCH₂CH₂N(CH₃)₂CH₂—), 4.28 (s, 2H, —CH₂N(CH₃)₂CH₂COO—), 4.60(t, J=3 Hz, 2H, CH₂═C(CH₃)COOCH₂CH₂N(CH₃)₂CH₂—), 5.73 and 6.10 (s, 2H,CH₂═C(CH₃)COO—).

NHS-PCB-tBu.

ATRP of CB-tBu was carried out in anhydrous dimethylformamide (DMF)using a Cu(I)Br/HMTETA catalyst. In a typical polymerization DMF and theliquid HMTETA ligand are separately purged of oxygen by bubbling withnitrogen. 1 g (3.67 mmol) of CB-tBu monomer and 125 mg (0.5 mmol) ofNHS-initiator were added to a Schlenk tube. To a second Schlenk tube wasadded 71.7 mg (0.5 mmol) of Cu(I)Br. Both tubes were deoxygenated bycycling between nitrogen and vacuum three times. 8 and 2 mL ofdeoxygenated DMF were added to the monomer/initiator and Cu(I)Br tubes,respectively. 136 μL (0.50 mmol) of deoxygenated HMTETA was added to theCu(I)Br containing solution and was stirred for 30 min under nitrogenprotection. The catalyst solution (Cu(I)/HMTETA) was then all added tothe monomer/initiator solution to start the reaction. The reaction wasrun overnight at room temperature. After polymerization, the reactionwas fully precipitated in ethyl ether. The precipitate was then driedunder vacuum and redissolved in minimal DMF (3-5 mL). This solution wasvortexed until fully dissolved and precipitated in acetone to remove thesoluble catalyst and trace monomer. This was repeated for a total of 3times to fully remove the catalyst. The remaining ester polymer wasdried overnight under vacuum and analyzed by NMR. (754 mg, yield=74.5%).

NHS-PCB and the Molecular Weight Measurement.

NHS-PCB was obtained by hydrolysis of tBu Groups. 500 mg NHS-PCB-tBu wasdissolved in 5 ml trifluoroacetic acid. This was allowed to sit for 2hours. The solution was then precipitated in ethyl ether, driedovernight under vacuum. Molecular weight (e.g., around 4,909 Da) wasdetermined from ¹H NMR (D₂O) δ (ppm): 2.82 (s, 4H, —COCH₂CH₂CO— from theinitiator), 3.27 (b, 6H, —CH₂N(CH₃)₂CH₂COO⁻ from CBMA-1), 4.2 and 4.5(m, 6H, —COOCH₂CH₂N(CH₃)₂CH₂COO⁻). Note that free NHS will have ¹H NMR(D₂O) δ (ppm): 2.67 (s, 4H, —COCH₂CH₂CO— from the initiator). Waterphase GPC also shows MW=5410 Da with PDI=1.03. These MW data were usedto characterize the MW for PCB block in DSPE-PCB.

DSPE-PCB.

1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE, GenzymePharmaceuticals) was used as received. ¹H NMR (trifluoroacetic acid-d) δ(ppm): 0.907 (6H, t, J=6.3 Hz, —CH₃), 1.344 (56H, br s, CH₃—(CH₂)₁₄—),1.752 (4H, m, CH₃—(CH₂)₁₄—CH₂—,), 2.551 (4H, m, CH₃—(CH₂)₁₅—CH₂—), 3.664(2H, s, —CH₂—NH₃ ⁺), 4.438 and 4.606 (6H, m,—CH₂—CH(C₁₇H₃₅COO)—CH₂—PO₄—CH₂—), 5.581 (1H, m, sn2-CH).

For a typical reaction, 120 mg NHS-PCB-tBu (MW for PCB is 5K) and 120 mgDSPE were stirred in 30 ml chloroform/4.3 ml DMF mixed solvents in thepresence of 129 μl triethylamine for days. The reaction mixture was thenevaporated and precipitated in ethyl ether. The precipitates was firstextracted by acetonitrile and filtered. The filtrate was evaporated,treated by TFA for 4 h, precipitated in ethyl ether and vacuum dried.The dry product was neutralized in 200 mM phosphate buffer (pH=8) with20 mM hydroxylamine, and gone through ultrofiltration (30K MW cutoff) inPBS followed in water repeatedly. Pure DSPE-PCB conjugates were retainedin this process and freeze-dried. (yield=58%).

The formation of DSPE-PCB conjugation was confirmed and the molar ratio(DSPE/CB) was determined to be 1/20 by ¹H NMR (trifluoroacetic acid-d) δ(ppm): 2.567 (m, 4H of CH₃—(CH₂)₁₅—CH₂— from DSPE), 4.637 (br m, 6H of—CH₂—CH(C₁₇H₃₅COO)—CH₂—PO₄—CH₂— from DSPE, and 6H of—COOCH₂CH₂N(CH₃)₂CH₂COO⁻ from PCB), 5.590 (s, 1H of sn2-CH from DSPE).This 1/20 (DSPE/CB) molar ratio implies roughly 5000 Da PCB conjugatedto one DSPE molecule, in agreement with the molecular weight ofunconjugated PCB determined via NMR and GPC methods.

DSPE-PCB Liposomes

In a typical formulation, lipid components (e.g., including DSPC andDSPE-PCB or commercial DSPE-PEG) were mixed in 2,2,2-trifluoroethanol atdesired molar ratio. A thin lipid film was formed by evaporation.Hydration of the lipid film was achieved by addition of PBS at 60° C.followed by 3 freeze-thaw cycles. The resulting liposomes were extruded20 times through Avanti® Mini-Extruder at 60° C. equipped with apolycarbonate membrane (80 nm). Particle sizes and its polydispersityindexes (PDI) were measured in PBS, and zeta-potentials were measured inwater. Typical liposome sizes and zeta-potentials for variedformulations are shown in FIGS. 5A-5D. Generally these liposomes werearound 100 nm with low PDI numbers. These liposomes were analyzedthrough size exclusion chromatography (SEC) by a Waters Alliance 2695Separations Module equipped with a Waters 2414 refractive index detectorand a custom-built size exclusion column (Tricorn™ 10/600 Column packedwith Sephacryl™ S-500 HR chromatography medium, GE Healthcare,Piscataway, N.J., USA). The mobile phase was PBS solution at a flow rateof 1 ml/min at 25° C. It should be noted that both DSPE-PCB and DSPE-PEGtested will form micelles alone in aqueous solution. A comparison ofFIGS. 5A and 5B (DSPE-PCB) to FIGS. 5C and 5D (DSPE-PEG) indicates thatwithin the same molecular weight, DSPE-PCB was able to incorporate moreinto the liposomes without leading to micellization. Liposomes wereincubated in PBS at 37° C. to test their stability from aggregation.FIG. 6 shows only DSPE-PCB and DSPE-PEG conjugate modified liposomeswere able to protect liposomes from long-term aggregation, whileformulations lacking these conjugates started aggregating within fewhours.

TABLE 1 Size and zeta-potential properties for different formulations.Liposomes Mean Diameter Zeta-potential (mol/mol) (nm) PDI (mV)DSPC/DSPE-PCB 139.9 ± 1.778 0.084 ± 0.012  −41.0 ± 0.100 5K (9/0.5)DSPC/DSPE-PCB 127.5 ± 0.781 0.159 ± 0.008 −42.7 ± 1.50 5K (9/1)DSPC/DSPE-PCB  84.86 ± 0.4167 0.185 ± 0.007 −40.6 ± 1.31 5K (9/1.5)DSPC/DSPE-PCB 91.27 ± 4.456 0.284 ± 0.008  −38.0 ± 0.651 5K (9/2)DSPC/DSPE-PCB 115.3 ± 3.322 0.145 ± 0.033 −32.9 ± 3.68 2K (9/0.5)DSPC/DSPE-PCB 103.2 ± 2.997 0.121 ± 0.033 −43.3 ± 3.11 2K (9/1)DSPC/DSPE-PCB 104.9 ± 1.223 0.084 ± 0.017 −43.4 ± 1.14 2K (9/1.5)DSPC/DSPE-PCB  122.6 ± 0.9252 0.132 ± 0.027 −48.6 ± 2.40 2K (9/2)DSPC/DSPE-PEG 160.4 ± 3.444 0.021 ± 0.013 −20.3 ± 3.61 5K (9/0.5)DSPC/DSPE-PEG  122.2 ± 0.7234 0.239 ± 0.012 −16.5 ± 1.74 5K (9/1)DSPC/DSPE-PEG 92.81 ± 3.010 0.362 ± 0.014  −16.5 ± 0.603 5K (9/1.5)DSPC/DSPE-PEG  96.14 ± 0.5859 0.401 ± 0.009  −13.8 ± 0.252 5K (9/2)DSPC/DSPE-PEG 132.0 ± 6.622 0.195 ± 0.018  −26.9 ± 0.451 2K (9/0.5)DSPC/DSPE-PEG 129.2 ± 5.027 0.083 ± 0.075 −31.8 ± 1.40 2K (9/1)DSPC/DSPE-PEG 136.3 ± 10.21 0.129 ± 0.103  −33.2 ± 0.404 2K (9/1.5)DSPC/DSPE-PEG 102.5 ± 6.271 0.280 ± 0.031 −32.3 ± 1.46 2K (9/2)DSPC/DSPE-PEG 121.0 ± 4.709 0.296 ± 0.047  −30.8 ± 0.964 2K (9/2.5) 5Kor 2K represents the MW for the PCB or PEG polymers. Data were measuredby triplicates and illustrated as mean value ± standard deviation.

In Vivo Circulation Studies on DSPE-PCB Liposomes.

All the liposomes listed in Table 2 are labeled fluorescently by adding1 mol % 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissaminerhodamine B sulfonyl) (Avanti) during the liposome formulationprocesses. All these liposomes formulation are below 150 nm with nearlymonopolydispersity. These formulations are evaluated on rats for theircirculation profiles. Sprague Dawley (150 g) rats were randomly dividedinto groups (group size is 3) and anesthetized with 3-5% isofluran. 200μl of each liposome formulation containing 2.3 μmol phospholipids wasinjected through the tail vein. At 5 min, 4 h, 8 h, 24 h, and 48 h, therat was anesthetized with 3-5% isofluran temporarily and 50-100 μl bloodwas drawn from either left or right sophenous vein. After 48 h, ratswere euthanized by CO₂ inhalation. The blood collected will be weightedand mixed with water/acetonitrile cocktails. After brief centrifugation,the supernatants will be measured for fluorescent emission at 583.6 nmwith the excitation at 557 nm. Calibration curves were established toquantify the doses of liposomes in these blood samples. The dose datavs. the time were fitted by a one-compartment pharmacokinetic model andthe circulation half-life and area under the curve (AUC) data werecalculated and shown in Table 3. Significant longer circulation ofDSPE-PCB liposomes was observed than DSPE-PEG counterparts. Such asDSPC/DSPE-PCB 5K (9/1) has a half-life of 9-10 hours, much longer thanDSPC/DSPE-PEG 5K (9/1) which is 3-4 hours.

TABLE 2 Size and zeta-potential properties for different formulations.Liposomes Formulation Mean Diameter Zeta-potential (mol/mol) (nm) PDI(mV) DSPC 119.3 ± 3.474 0.105 ± 0.015 −18.0 ± 2.26  DSPC/DSPE-PCB  144.0± 0.6245 0.106 ± 0.016 −45.3 ± 0.577 2K (9/1) DSPC/DSPE-PEG  127.8 ±0.7095 0.135 ± 0.011 −35.9 ± 0.173 2K (9/1) DSPC/DSPE-PCB 112.6 ± 2.7560.062 ± 0.023 −46.8 ± 0.808 2K (9/1.5) DSPC/DSPE-PEG 97.38 ± 2.847 0.081± 0.013 −32.5 ± 1.81  2K (9/1.5) DSPC/DSPE-PCB 104.9 ± 2.489 0.053 ±0.019 −36.8 ± 0.306 5K (9/0.47) DSPC/DSPE-PEG 113.6 ± 4.284 0.041 ±0.014 −16.8 ± 0.985 5K (9/0.47) DSPC/DSPE-PCB 98.31 ± 1.442 0.087 ±0.009 −37.5 ± 0.300 5K (9/1) DSPC/DSPE-PEG 120.6 ± 2.169 0.063 ± 0.015−17.4 ± 1.10  5K (9/1) 5K or 2K represents the MW for the PCB or PEGpolymers. Data were measured by triplicates and illustrated as meanvalue ± standard deviation.

TABLE 3 In vivo circulation profiles for different formulations.Liposome Formulation (mol/mol) Half-Life (h) AUC (% ID h) DSPC 0.70 ±0.04 108.73 ± 5.97  DSPC/DSPE-PCB 2K (9/1) 3.09 ± 0.10 462.28 ± 14.67DSPC/DSPE-PEG 2K (9/1) 2.25 ± 0.10 330.46 ± 12.97 DSPC/DSPE-PCB 2K(9/1.5) 6.60 ± 0.62  982.82 ± 104.92 DSPC/DSPE-PEG 2K (9/1.5) 4.05 ±0.18 588.75 ± 26.54 DSPC/DSPE-PCB 5K (9/0.47) 5.55 ± 0.30 830.90 ± 51.31DSPC/DSPE-PEG 5K (9/0.47) 4.36 ± 0.06 633.51 ± 11.63 DSPC/DSPE-PCB 5K(9/1) 9.56 ± 1.58 1489.07 ± 276.98 DSPC/DSPE-PEG 5K (9/1) 3.74 ± 0.23534.84 ± 34.20 5K or 2K represents the MW for the PCB or PEG polymers.Data were measured by triplicates and illustrated as mean value ±standard deviation. % ID represents % injected dose.

Example 2 Preparation and Characteristics of a RepresentativeNanoparticle Drug Delivery System: Amphiphilic PLGA Zwitterionic BlockCopolymers

In this example, the preparation and characteristics of a representativenanoparticle drug delivery system of the invention are described.

Materials.

2-bromoisobutyryl bromide, t-Boc-aminoethyl alcohol,2-(dimethylamino)ethyl methacrylate, tert-butyl bromoacetate, Cu(I)Br,1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC),2,2,2-trifluoroethanol (TFE), and 4-aminophenyl β-D-galactopyranoside(NH2-galactose) were purchased from Sigma-Aldrich, St. Louis, Mo.Trifluoroacetic acid (TFA) and N-hydroxysuccinimide (NHS) were purchasedfrom Acros Organics USA, Morris Plains, N.J.Poly(D,L-lactide-co-glycolide) (PLGA) with a 50:50 monomer ratio werepurchased from Durect Corporation, Pelham, Ala. Docetaxel (Dtxl) waspurchased from LC Laboratories, Woburn, Mass. 5-(Aminomethyl)fluoresceinhydrochloride, and22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol(NBD) were purchased from Invitrogen, Carlsbad, Calif.

Nanoparticle Preparation

2-Aminoethyl 2-Bromoisobutyrate.

The ATRP initiators with NH₂ functional groups were synthesized asfollows. Briefly, 3.57 g 2-bromoisobutyryl bromide was added to asolution of 2.5 g t-Boc-aminoethyl alcohol and 1.73 g triethylamine in 8ml methylene chloride in an ice bath. After 4 h reaction, the salts werefiltered off and the filtrate was extracted with saturated sodiumbicarbonate solution. Methylene chloride phase was dried over magnesiumsulfate and evaporated. The resulting t-Boc-aminoethyl2-bromoisobutyrate was treated by 15 ml trifluoroacetic acid (TFA) for 2h and crystallized upon addition of ethyl ether (yield 95%). ¹H NMR(DMSO-d₆) δ (ppm): 1.93 (s, 6H, —C(CH₃)₂Br), 3.16 (s, 2H, TFA⁻.NH₃⁺CH₂CH₂OCO), 4.31 (t, J=5 Hz, 2H, TFA⁻.NH₃ ⁺CH₂CH₂OCO), 8.22 (s, 3H,TFA⁻.NH₃ ⁺CH₂CH₂OCO).

2-tert-butoxy-N-(2-(methacryloyloxy)ethyl)-N,N-dimethyl-2-oxoethanaminium(CB-tBu monomer)

5 g 2-(Dimethylamino)ethyl methacrylate and 8.68 g tert-butylbromoacetate were reacted in 20 ml acetonitrile for 24 h at 50° C. underN₂ protection. Upon addition of 250 ml ethyl ether to the reactionmixture, the formed white crystals were isolated and dried. Theresulting CB-tBu monomers were immediately stored in a desiccator at−20° C. (yield 96%). ¹H NMR (D₂O) δ (ppm): 1.44 (s, 9H, —OC(CH₃)₃), 1.87(s, 3H, CH₂═C(CH₃)COO—), 3.31 (s, 6H, —CH₂N(CH₃)₂CH₂COO—), 3.98 (t, J=3Hz, 2H, CH₂═C(CH₃)COOCH₂CH₂N(CH₃)₂CH₂—), 4.28 (s, 2H,—CH₂N(CH₃)₂CH₂COO—), 4.60 (t, J=3 Hz, 2H,CH₂═C(CH₃)COOCH₂CH₂N(CH₃)₂CH₂—), 5.73 and 6.10 (s, 2H, CH₂═C(CH₃)COO—).

Synthesis of PCB-tBu Polymers.

The ATRP of CB-tBu monomers was carried out as follows: 74 mg Cu(I)Brand 148.6 mg HMTETA were placed into a Schlenk tube and underwent threevacuum-nitrogen cycles. Then, 7 ml degassed DMF was added to makesolution A. Similarly, 1.8 g CB-tBu monomers and 80 mg 2-aminoethyl2-bromoisobutyrate were placed into another Schlenk tube with oxygenfully excluded, followed by addition of 8 ml degassed DMF to makesolution B. Polymerization was started by transferring solution B intosolution A under N₂ protection. After reaction at 60° C. for 24 h, thepolymers were first precipitated in ethyl ether, then were dissolved ina small amount of ethanol and precipitated in acetone repeatedly toremove residual monomers, initiators and catalysts. The resultingPCB-tBu was dried under vacuum before further use.

PLGA-b-PCB-tBu Conjugation.

The conjugation process was via NHS/EDC chemistry. Briefly, 3.2 g COOHterminated PLGA (0.20 dl/g), 86.4 mg NHS and 147.2 mg EDC were reactedin 6 ml methylene chloride for 4 h at room temperature. Then, 5 ml ethylether was added to obtain white precipitates. The resulting PLGA-NHS waswashed with cold ethyl ether/methanol mixture (2/1, v/v) to remove anyNHS and EDC residuals, then vacuum-dried before use. TFA⁻.NH₃⁺-terminated pCBMA-tBu was treated with an excess of triethylamine toremove TFA protection. NH₂-terminated pCBMA-tBu was purified viafiltration, precipitated into ethyl ether, and vacuum-dried. 878 mgNH₂-terminated PCB-tBu and 1.68 g PLGA-NHS were conjugated in thepresence of 50 μl triethylamine in 7 ml acetonitrile at 60° C. for 20 h.The resulting PLGA-PCB-tBu was precipitated in cold methanol. PCBMA-tBucontaminant was removed by repeating the washing cycle. The formation ofPLGA-PCBMA-tBu conjugation was confirmed and the weight ratio (PLGA/PCB)was determined to be 6/1 (mole ratio 12.4/1) by ¹H NMR (Acetonitrile-d₃)δ (ppm): 1.55 (m, 3H, —COCH(CH₃)O—, in PLGA, and 9H, —OC(CH₃)₃ inPCB-tBu), 3.65 (br, 6H, —CH₂N(CH₃)₂CH₂COOC(CH₃)₃, in PCB-tBu), 4.85 (m,2H, —COCH₂O—, in PLGA), 5.22 (m, 1H, —COCH(CH₃)O—, in PLGA). Thepolymers were dried in vacuum before use.

Hydrolysis of tBu Ester Groups.

In control experiments, PLGA treated with TFA for up to 6 h did not showsignificant molecular weight changes, and PCB-tBu after 1 h TFAtreatment showed no signal at 1.44 ppm in ¹H NMR (D₂O) indicating thattBu ester groups are fully removed. To confirm that ester bonds atmethacrylates were not destroyed following the TFA treatment, CB-tBumonomers were hydrolyzed in TFA for 1 h and the hydrolyzed productidentified as CB zwitterionic monomers by ¹H NMR (D₂O) δ (ppm): 1.89 (s,3H, CH₂═C(CH₃)COO—), 3.31 (s, 6H, —CH₂N(CH₃)₂CH₂COO—), 3.98 (t, J=3 Hz,2H, CH₂═C(CH₃)COOCH₂CH₂N(CH₃)₂CH₂—), 4.48 (s, 2H, —CH₂N(CH₃)₂CH₂COO⁻),4.54 (s, 2H, CH₂═C(CH₃)COOCH₂CH₂N(CH₃)₂CH₂—), 5.75 and 6.06 (s, 2H,CH₂═C(CH₃)COO—). To obtain PLGA-co-PCB, PLGA-co-PCB-tBu was treated withTFA for 1 h to remove tBu ester groups. The resulting PLGA-co-PCB wasprecipitated into ethyl ether, and re-dissolved in a small amount of TFEand precipitated in ethyl ether repeatedly. After vacuum dry, thecopolymers are ready for NP formulation. Weight ratio (PLGA/PCB) wasdetermined to be 10/1 by ¹H NMR (trifluoroacetic acid-d) δ (ppm): 5.50(m, 1H, —COCH(CH₃)O—, in PLGA), 5.10 (m, 2H, —COCH₂O—, in PLGA), 1.77(d, 3H, —COCH(CH₃)O—, in PLGA), 3.64 (br, 6H, —CH₂N(CH₃)₂CH₂COO—, inPCB).

The molecular weight and distribution of PCB homopolymers (derived byhydrolysis of PCB-tBu polymers) are determined by a Waters Alliance 2695Separations Module equipped with a Waters Ultrahydrogel 1000 column anda Waters 2414 reflex detector. The mobile phase was 100 mM NaCl aqueoussolution at a flow rate of 0.7 ml/min at 35° C. Poly(ethylene oxide)from Polymer Laboratories were used as standards. Gel permeationchromatography shows a molecular weight (Mn) of 13640 withpolydispersity of 1.12.

Formulation of PLGA-PCB NPs.

Solvent displacement (nanoprecipitation) method was used to formulateNPs. PLGA-PCB copolymers were dissolved in TFE/MeOH 1/1 v/v cosolvent atthe concentration of 0.5 mg/ml. Water (water: organic solvent volumeration, 4:1) was transferred to copolymer solution in a dropwise mannerunder 1000 rpm stir. After 2 h, solvents were exchanged to PBS andresulting PLGA-PCB NPs were concentrated to the desired concentration byan Amicon Ultra-4 centrifugal filter (Millipore, Billerica, Mass., US)with 100,000 Da MW cutoff. PLGA NPs were prepared as described in J.Cheng, B. A. Teply, I. Sherifi, J. Sung, G. Luther, F. X. Gu, E.Levy-Nissenbaum, A. F. Radovic-Moreno, R. Langer, o. C. Farokhzad,Biomaterials 2007, 28, 869. Docetaxel was loaded in the NPs by mixingwith polymers in the organic solvent and followed the above-mentionedprocedures to formulate PLGA-PCB/Dtxl NPs. The mean diameter,polydispersity index (PDI) and zeta-potential of NPs were determined byZetasier Nano-ZS (Malvern Instruments Ltd, Malvern, WR, UK) intriplicates. PDI ranging from 0 to 1.00 was used to characterize the NPsize distribution. NPs are considered as monodisperse when PDI<0.10.

Drug Loading and Releasing Kinetics.

After nanoprecipitation of polymers (either PLGA or PLGA-PCB) withdocetaxel, preparation solution containing drug-loaded NPs and freedrugs went through a Microcon centrifugal filter (Millipore, Billerica,Mass.) with 100,000 Da MW cutoff. The drug content in the filtrates(containing free drugs) along with the preparation solution werecompared to determine the drug loading (drug/polymers, w/w). In drugreleasing studies, Dtxl-loaded NP solutions were placed intoSlide-A-Lyzer MINI dialysis microtubes (3500 Da MW cutoff, Pierce,Rockford, Ill.) at 100 μl (0.33 mg/ml) per tube. Those tubes weredialyzed against 1 L PBS at 37° C. with gentle stirring. PBS wasrefreshed every 24 h. At varied time points, three microtubes were takento determine drug content retained by the NPs. All aqueous samples weremixed with equal-volume acetonitrile overnight to fully release the drugbefore running HPLC. Docetaxel content was quantified in triplicates bya Waters Alliance 2695 Separations Module equipped with a reverse-phaseC18 column (Econosil, 250×4.6 mm, 5 μm, Alltech, Deerfield, Ill., USA)and a UV detector (wavelength of 227 nm). The mobile phase was water andacetonitrile (v/v 50/50) at a flow rate of 0.5 ml/min at roomtemperature. The retention time for free Doc was 13.75 min.

PLGA-PCB NP Functionalization with Dye Molecules and Targeting Ligands.

Molecules of interest can be immobilized onto PLGA-PCB NPs via EDC/NHSchemistry. To conjugate dye molecules, NPs were incubated with 400 mMEDC and 200 mM NHS in water for 20 min, and washed with pure water toremove unreacted EDC and NHS. 0.5 mg NHS-activated NPs in 100 μl waterwere reacted with 25 μl of 5-(aminomethyl)fluorescein hydrochloride(Invitrogen, Carlsbad, Calif., US) at the concentration of 10 mg/ml in10 mM sodium borate buffer, pH=9 at dark place for 2.5 h. The resultingdye-NP conjugates were washed with pH 9 buffer and water, resuspended inwater, and analyzed with the FACScan flow cytometer (Becton Dickinson,San Jose, Calif.) at the concentration of 0.5 mg/ml. For theimmobilization of targeting ligands,22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol(NBD) was formulated into PLGA-PCB NPs with initial drug loading of 0.25wt %. The resulted PLGA-PCB/NBD NPs were activated by 400 mM EDC and 200mM NHS in water for 20 min. 0.5 mg NHS-activated NPs in 100 μl waterwere reacted with 10 μl of NH₂-galactose at the concentration of 25mg/ml in 10 mM sodium borate buffer, pH=9 for 1 h. The resultinggalactose-NP conjugates were washed with pH 9 buffer and PBS, andresuspended in PBS before cell incubation. HepG2 cells were grown in24-well plates in full MEM medium (Hyclone, Logan, Utah) supplementedwith non-essential amino acid, sodium pyruvate and 10% fetal bovineserum (FBS) under 5% CO₂ at 37° C. to allow 50% confluence. Cells werethen washed with pre-warmed PBS and incubated with 400 μl/wellNP-containing MEM medium without FBS supplement (PLGA-PCB/NBD-GalactoseNP concentration: 1.25 mg/ml). After 2 h, cells were washed with PBS andsupplemented with 400 μl/well FBS-containing medium. After 20 hincubation at 37° C., cells were visualized using a Nikon TE2000 Umicroscope. For control experiments, EDC was absence while all otherconditions and procedures were exactly the same.

Cytotoxicity Assays for PLGA-PCB NPs.

The cytotoxicity of the NPs was evaluated using a Vybrant® MTT CellProliferation Assay Kit (Molecular Probes, Eugene, Oreg.). Briefly,HepG2 cells were grown in 96-well plates in full MEM medium supplementedwith non-essential amino acid, sodium pyruvate and 10% FBS under 5% CO₂at 37° C. to allow 80-90% confluence. For each well, cells were washedwith PBS and incubated with 200 μl full medium containing variedconcentration of either PLGA-PCB or PLGA NPs for 24 h. Cells were washedwith PBS to remove NPs and incubated with 100 μl full medium plus 50 μlof 12 mM MTT stock solution for another 4 h. Then, MTT-containing mediumwas replaced with 150 μl DMSO to dissolve the formed crystal at 37° C.for 10 min. Absorbance (Abs) was measured at 570 nm using a SpectraMaxM5 microplate reader (Molecular Devices, Sunnyvale, Calif.) with pureDMSO as the blank reading. Cells with no NP incubation were used as thecontrols and cell viability upon NPs treatment was estimated intriplicate: cell viability (%)=Abs_(sample)/Abs_(control)×100.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

1. A block copolymer, comprising: (a) a zwitterionic polymer blockcomprising a poly(carboxybetaine); and (b) a hydrophobic block, whereinthe zwitterionic polymer block comprises a plurality of repeating units,each repeating unit having the formula:

wherein R₁ is selected from the group consisting of hydrogen, fluorine,trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups; R₂ and R₃ areindependently selected from the group consisting of alkyl and aryl, ortaken together with the nitrogen to which they are attached form acationic center; L₁ is a linker that covalently couples the cationiccenter [N⁺(R₅)(R₆)] to the polymer backbone [—(CH₂—CR₄)_(n)—]; L₂ is alinker that covalently couples the anionic center [A(═O)—O⁻] to thecationic center; A is C; M⁺ is a counter ion associated with the (A=O)O⁻anionic center; X⁻ is a counter ion associated with the cationic center;and n is an integer from 1 to about 10,000.
 2. The copolymer of claim 1,wherein R₁-R₈ are independently selected from the group consisting ofC1-C3 alkyl.
 3. The copolymer of claim 1, wherein L₃ is selected fromthe group consisting of —C(═O)O—(CH₂)_(n)— and —C(═O)NH—(CH₂)_(n)—,wherein n is 1-20.
 4. The copolymer of claim 1, wherein L₄ is—(CH₂)_(n)—, where n is an integer from 1-20.
 5. The copolymer of claim1, wherein n is an integer from 10 to about 1,000.
 6. The copolymer ofclaim 1, wherein p is an integer from 10 to about 1,000.
 7. A blockcopolymer, comprising: (a) a mixed charge copolymer block comprising amixed charge copolymer; and (b) a hydrophobic block, wherein the mixedcharge copolymer comprises a plurality of repeating units, eachrepeating unit having the formula:

wherein R₄ and R₅ are independently selected from hydrogen, fluorine,trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups; R₆, R₇, and R₈ areindependently selected from alkyl and aryl, or taken together with thenitrogen to which they are attached form a cationic center; A(═O)—OM) isan anionic center, wherein A is C, S, SO, P, or PO, and M⁺ is a metal ororganic counterion; L₃ is a linker that covalently couples the cationiccenter [N⁺(R₆)(R₇)(R₈)] to the polymer backbone; L₄ is a linker thatcovalently couples the anionic center [A(═O)—OM] to the polymerbackbone; X⁻ is the counter ion associated with the cationic center; nis an integer from 1 to about 10,000; and p is an integer from 1 toabout 10,000.
 8. The copolymer of claim 7, wherein R₁-R₈ areindependently selected from the group consisting of C1-C3 alkyl.
 9. Thecopolymer of claim 7, wherein L₃ is selected from the group consistingof —C(═O)O—(CH₂)_(n)— and —C(═O)NH—(CH₂)_(n)—, wherein n is 1-20. 10.The copolymer of claim 7, wherein L₄ is —(CH₂)_(n)—, where n is aninteger from 1-20.
 11. The copolymer of claim 7, wherein n is an integerfrom 10 to about 1,000.
 12. The copolymer of claim 7, wherein p is aninteger from 10 to about 1,000.
 13. A zwitterionic conjugate, comprisinga lipid covalently coupled to a poly(carboxybetaine), apoly(sulfobetaine), or a poly(phosphobetaine), wherein thepoly(carboxybetaine), poly(sulfobetaine), or poly(phosphobetaine)comprises a plurality of repeating units, each repeating unit having theformula:

wherein R₁ is selected from the group consisting of hydrogen, fluorine,trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups; R₂ and R₃ areindependently selected from the group consisting of alkyl and aryl, ortaken together with the nitrogen to which they are attached form acationic center; L₁ is a linker that covalently couples the cationiccenter [N⁺(R₅)(R₆)] to the polymer backbone [—(CH₂—CR₄)_(n)—]; L₂ is alinker that covalently couples the anionic center [A(═O)—O] to thecationic center; A is C, S, SO, P, or PO; M⁺ is a counter ion associatedwith the (A=O)O⁻ anionic center; X⁻ is a counter ion associated with thecationic center; and n is an integer from 1 to about 10,000.
 14. Theconjugate of claim 13, wherein the lipid is distearoylphosphatidylethanolamine (DSPE).
 15. A mixed charge copolymer conjugate,comprising a lipid covalently coupled to a mixed charge copolymer,wherein the mixed charge copolymer comprises a plurality of repeatingunits, each repeating unit having the formula:

wherein R₄ and R₅ are independently selected from hydrogen, fluorine,trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups; R₆, R₇, and R₈ areindependently selected from alkyl and aryl, or taken together with thenitrogen to which they are attached form a cationic center; A(═O)—OM) isan anionic center, wherein A is C, S, SO, P, or PO, and M⁺ is a metal ororganic counterion; L₃ is a linker that covalently couples the cationiccenter [N⁺(R₆)(R₇)(R₈)] to the polymer backbone; L₄ is a linker thatcovalently couples the anionic center [A(═O)—OM] to the polymerbackbone; X⁻ is the counter ion associated with the cationic center; nis an integer from 1 to about 10,000; and p is an integer from 1 toabout 10,000.
 16. The conjugate of claim 15, wherein the hydrophobicmoiety is distearoyl-phosphatidylethanolamine (DSPE).
 17. An assembly,comprising a plurality of block copolymers of claim
 1. 18. The assemblyof claim 17 in the form of a core-shell polymeric particle.
 19. Anassembly, comprising a plurality of conjugates of claim
 13. 20. Theassembly of claim 19 in the form of a micelle, a liposome, or apolymersome.
 21. A composition, comprising the assembly of claim 17 anda pharmaceutically accepted carrier or diluent.